The regulation of intracellular iron is a major cellular homeostasis problem. Tightly controlled regulatory mechanisms must be present in the cell to prevent the deleterious effects of iron starvation or iron toxicity. This homeostatic problem is evident at the organism level, where iron deficiency causes anemia, while an excess of iron results in hemochromatosis, both serious cardiovascular diseases. In Saccharomyces cerevisiae, genes encoding iron transporters and iron uptake facilitators are activated by the transcription factors Aft1/2p in low iron conditions. In particular, Aftlp activates the FIT family of proteins that facilitate the uptake of siderophore-bound iron through the cell wall. Previous work from our lab has shown that in normal iron conditions, FIT3 mRNA is highly expressed in cells lacking yeast RNase III and the presence of a 5'extended species in cells deficient in the exoribonucleases Xrn1 and Rat1. The 5'end of these extended species were found to originate ~3.3kb upstream of the FIT3 translation initiation codon. Clearly, the regulation of FIT3 involves mechanisms other than induction by the transcriptional activators Aft1/2p. We propose to study these novel mechanisms of FIT3 regulation associated with cellular iron homeostasis using the yeast Saccharomyces cerevisiae. We are testing the hypothesis that the transcription of the FIT3 gene is repressed in normal iron conditions by transcriptional interference from an upstream promoter. It is unknown whether this upstream promoter is regulated in normal/low iron conditions, which in turn regulates the downstream FIT3 promoter. Our efforts in elucidating the role of transcriptional interference in the regulation of the FIT3 gene will be focused on analyzing the promoter region upstream of the FIT3 gene that gives rise to these 5'extended RNA species. We will also investigate FIT3 repression by upstream non-coding RNAs and the transcriptional regulation of these upstream non-coding RNAs. Lastly, we will identify activator/represser proteins involved in the regulation of the upstream promoter and FIT3 promoter in conditions of low iron. Elucidating the transcriptional regulation of genes involved in iron homeostasis in yeast is important because it could identify regulatory paradigms and novel regulatory proteins, which might be conserved in pathogenic fungi or mammalian iron homeostasis. Our studies should identify novel regulatory proteins and possible regulatory mechanisms that may be conserved in pathogenic yeast fungi and prompt future studies in the development of antifungal therapies, as iron is vital to all known fungi.